Project Summary/Abstract Structure and Mechanism of Class II Aminoacyl-tRNA Synthetases All cells require protein synthesis to maintain their core physiology, as well as permit orderly cell division and regulated differentiation. Mutations and expression changes in enzymes of the translation apparatus can present with complex phenotypes in human diseases, representing a major gap in knowledge. The limited tissue and organ specific nature (e,g, the brain) of these diseases is also a challenge. The long term objective of this work is to discover how specific changes, both genetic and at the level of expression, in the function of conserved components of the translational apparatus bring about human disease. A better understanding of this gene phenotype relationship as it relates to translation factors will have major implications for human health. The immediate goal of this application is to understand how two aminoacyl-tRNA synthetases- histidyl- tRNA synthetase (HARS) and threonyl-tRNA synthetase (TARS)- undergo functional switching between their canonical role in protein synthesis and secondary roles in other physiological processes. Alterations in this switching in HARS and TARS may be linked to Type IIIb Usher syndrome and cancer, respectively. Here, we will test specific hypotheses to account for these secondary functions. For HARS and the Type IIIb Usher syndrome, we hypothesize that the mutant substitution linked to the disease decreases histidyl-tRNAHis levels, leading to near codon suppression or nutritional deficit dependent stress responses. Alternatively, protein- protein interactions or signaling pathways associated with a previously undiscovered secondary function may be perturbed. For TARS, we hypothesize that its association with cancer requires a switch to a non-canonical angiogenesis related function and its subsequent export from cells via an exosome dependent pathway. The application contains two multi-part Aims: (1) Determine the molecular basis of the link between Y454S human histidyl-tRNA synthetase and type III Usher syndrome by (i) measuring the contribution of HisRS under-acylation/miscoding to the Usher IIIB phenotype; (ii) determining the extent to which Type IIIb Usher Syndrome is the result of a loss a HARS-protein protein interaction; and (iii) determining the association of Y454S HisRS with altered signaling by determining the phosphorylation state of HARS. (2) Determine the mechanism by which human threonyl-tRNA synthetase switches between its canonical aminoacylation function and its novel angiogenic function by (i) Determining how the intracellular location of TARS changes in response to drug, hypoxia, nutritional deficit, and cytokines that elicit secretion (TNF-a); (ii) determining the extent to which TARS secretion and pro-angiogenic functios are associated with exosomes; and (iii), determining the role of TARS's cryptic GTPase function in pro-angiogenic functions. The research is significant for the knowledge gained regarding the underlying basis of protein functional diversity and flexibility, and for the high likelihood that further insights into the mechanisms of sensorineural disease and cancer will be gained.